This overall aim of this proposal is to examine the properties of gap junction (GJ) channels in the CNS that are likely to be relevant to the operation of electrical synapses. GJ channels, composed of the molecularly diverse family of connexins, are the morphological substrates of direct cell-cell communication. The CNS expresses at least eleven different connexins and combined genetic and morphological studies have established that neurons express Cx36, astrocytes Cx43 and Cx30 and oligodendrocytes Cx32, Cx45 and Cx47. A new GJ gene, Cx30.2 also appears to be expressed in neurons. In this proposal, we will examine which combinations of these CNS connexins form channels, how they interact and how channel properties differ. We will use color variants of GFP-tagged connexins expressed in mammalian cells which allows us to combine electrophysiological recording and fluorescence imaging to examine functional coupling, junction formation and connexin interaction simultaneously in living cells. GJ communication in the CNS may also transmit metabolites and signaling molecules and we will focus on examining the permeability characteristics of Cx36 and the other putative neuronal connexin, Cx30.2. Of particular interest is the transmission of the biological signaling molecules lP3, cAMP and cGMP. Imaging combined with the use of cyclic nucleotide gated channels as biosensors will be used to detect intercellular fluxes of these molecules in living cells. Both electrical and chemical coupling in the CNS is dynamically modulated and in number of brain regions appears to be linked to second messenger cascades coupled to neurotransmitter receptors. Most often action involves cyclic nucleotides, but heterogeneity in connexin expression among diverse neuronal types has shown inconsistency of action. We will use a simplified system comprised of cells transfected with Cx36 or Cx30.2 in isolation to examine modulatory effects of cyclic nucleotides and extend these studies to investigate possible action through connexin phosphorylation. We will also investigate the effects of pH, which may act directly on connexins or indirectly through modulation of transmitter action. Finally, we take a step towards examining connexin expression in neurons by utilizing an immortalized hippocampal progenitor cell line that undergoes differentiation following treatment with defined factors. We will express GFP-tagged connexins in these cells and examine dynamics of connexin expression in processes and soma and changes in distribution that occur with differentiation, neuronal activity and initiation of cell-cell contact with other neurons and with gHa.